Figure 3. Time-dependent increases in tone responses of PVT neurons following fear conditioning.

(a) Diagram of recording placements in PVT. Coordinates from bregma. (b) Percentage of tone responsive (TR) neurons at 2 h and 24 h following conditioning (n= 54 neurons, 6% tone responsive at 2 h, 20% tone responsive at 24 h, Fisher exact test, P= 0.04). (c) Average peri-stimulus time histograms of all PVT neurons that were significantly tone responsive at either 2 h or 24 h after conditioning (n=13 neurons). (d) Maximum z-score for group data (bars) or individual data (gray lines, n=13 neurons). Dashed line indicates a z-score criterion of 2.58. (e) Changes in spontaneous firing rates of PVT neurons at 2 h (left) and 24 h (right) after conditioning, compared to pre-conditioning. Changes at 24 h were significantly greater than 2 h (n= 54 neurons; 24 h= 80%; 2 h= 57%, Fisher exact test, P= 0.02). (f_1,left) Micrograph showing the site of retrobeads infusion into PVT. (f₁, right) Micrograph showing PL neurons projecting to PVT (retrogradely labeled, red) expressing immunoreactivity for cFos (green) following fear retrieval at 7 d. Scale bar,100 μm. (f₂) Confocal images showing cFos labeling (left), retrobeads labeling (middle), and overlay (right) of PVT-projecting PL neurons (white arrows). Scale bar,10 μm. (f₃) Fear retrieval at 7 d increased the number of cFos positive neurons (per 0.1 mm²) in PL (left, P= 0.003, t= 8.74), but the number of retrogradely labeled PL neurons was the same between groups (middle, P= 0.51, t= −0.7). Retrieval increased the percentage of retrogradely labeled PL neurons expressing cFos (right, P= 0.004, t= 4.83; n=3–4 per group). (g_1,left) Micrograph showing the site of retrobeads infusion into CeA. (g_1,right) Micrograph showing PVT neurons projecting to CeA (retrogradely labeled, red) expressing immunoreactivity for cFos (green) after fear retrieval at 7 d. Scale bar,100 μm. (g₂) Confocal images showing cFos labeling (left), retrobeads labeling (middle), and overlay (right) of CeA-projecting PVT neurons (white arrow). Scale bar,10 μm. (g₃) Fear retrieval at 7 d increased the number of cFos positive neurons (per 0.1 mm²) in PVT (left, P= 0.03, t= 3.71), but the number of retrogradely labeled PVT neurons was the same between groups (middle, P= 0.79, t= −0.28). Retrieval increased the percentage of retrogradely labeled PVT neurons expressing cFos (right, P= 0.03, t= 3.61; n=2–3 per group). (h) Schematic of potential circuit mediating fear retrieval at 7d timepoint. Data are shown as mean ± SEM; Unpaired t-test between control and conditioned groups; *P < 0.05.